August/11 August 2009

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Revision as of 10:26, 11 August 2009 by Unocchi (Talk | contribs)

1.before Min prep
Checked the cell cultures transformed and plated out yesterday (8/10) for colony formation and made an approximate count of the number of colonies.

(plate number)-(location on plate) (no. of colonies)
1-23L    100>
1-15N    10
2-6P     10<
1-6I     10<
1-12H    10
1-18C    10<
1-20F    ×

inoculate to iquid medium(5mL + Amp 5uL or Kan 25uL)


2.digestion and ligation

digestion with restriction enzyme

Vector1
1-2M  12
SpeⅠ 1
PstⅠ 1
No.2 Buffer 2
dH2O 4
total 20uL

Insert1
2-8M   5
XbaⅠ  1
PstⅠ  1
No.2   2
dH2O   11
total  20 uL


Vector2
1-23L  12
EcoRⅠ 1
XbaⅠ  1
No.2   2
dH2O   4
total  20uL

Insert2
3-18O  5
EcoⅠ  1
SpeⅠ  1
No.2   2
dH2O   11
total  20uL

↓
37℃ , 2hr


gel electrophoresis
gel cut

purification by [QIAquick Nucleotide Removal Kit]

ligation

ligation
DNA        44
10* buffer 5
ligation   1
total      50uL
↓
16℃ overnight


3.Transformation
to get new plasmid

each 4uL(DNA) , 1-14F : 2uL
1-14H kan
1-14F kan
2-18F Amp ('8/10competent cell

1-23J Amp
1-12C Amp (super competent cell??